The environmental and host-associated bacterial microbiota of Arctic seawater-farmed Atlantic salmon with ulcerative disorders.

The Norwegian aquaculture of Atlantic salmon (Salmo salar L.) is hampered by ulcerative disorders associated with bacterial infections. Chronic ulceration may provide microenvironments that disturb the normal microbial biodiversity of external surfaces. Studying the composition of microbial communities in skin ulcers will enhance our understanding of ulcer aetiology. To achieve this, we tested marine farmed Atlantic salmon and sampled the base and edge of ulcers at the end of winter (April) and end of summer (September), in addition to skin mucus of healthy individuals. In order to assess microbiota associated with the host and obtain insight into the environmental ecology, we also sampled sea water, the sediment layer underneath the farm facility and the distal intestine of Atlantic salmon. The skin microbiota of Atlantic salmon was different from that of the surrounding water. Residential Tenacibaculum and Arcobacter species persistently dominated the cutaneous skin and ulcer mucus surfaces of Atlantic salmon during both winter and summer periods. The intestinal microbiota was dominated by Mycoplasma with an increase in Aliivibrio and Alcaligenes abundance in the intestine of fish with ulcerative disorder at the end of winter. These findings suggest the presence of resilient microbes in the mucus surfaces of Atlantic salmon.

Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence.

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.

Parameters affecting gene expression from the Pm promoter in gram-negative bacteria.

The Pm promoter inserted chromosomally or in broad-host-range replicons based on plasmid RSF1010 or RK2 are useful systems for both high- and low-level expression of cloned genes in several gram-negative bacterial species. The positive Pm regulator XylS is activated by certain substituted benzoic acid derivatives, and here we show that these effectors induce expression of Pm at similar relative ranking levels in both Escherichia coli and Pseudomonas aeruginosa However, the kinetics of expression was not the same in the two organisms. Different carbon sources and dissolved oxygen levels displayed limited effects on expression, but surprisingly the pH of the growth medium was found to be of major importance. By combining the effects of genetic and environmental parameters, expression from Pm could be varied over a ten-thousand- to a hundred-thousand-fold continuous range, and as an example of its applications we showed that Pm can be used to control the xanthan biosynthesis in Xanthomonas campestris.

Pm promoter expression mutants and their use in broad-host-range RK2 plasmid vectors.

By coupling the Pm/xylS promoter system to minimal replicons of the broad-host-range plasmid RK2 we recently showed that such vectors are useful for both high- and low-level inducible expression of cloned genes in gram-negative bacteria. In this report, we extend this potential by identifying point mutations in or near the -10 transcriptional region of Pm. Point mutations leading to gene-independent enhancements of expression levels of the induced state or reduced background expression levels were identified using Escherichia coli as a host. By combining these mutations an additive effect in expression levels from the constructed Pm was observed. The highest induced expression level was obtained by inserting an E. coli consensus sigma70 - 10 recognition region. Most of the remaining activities in the reduced-background mutations appeared to originate from a transcriptional start site other than Pm. The effects of some of these mutations were also analyzed in Pseudomonas aeruginosa and were found to act similarly, but less pronounced in this host.

Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.

The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment.

Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria.

This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatny et al., 1997). These vectors contain the minimal replicon of RK2 and the inducible Pu or Pm promoters together with their regulatory xylR or xylS genes, respectively, from the Pseudomonas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJB653, and these vectors are characterized by the relatively small size, the presence of multiple cloning sites downstream of Pm, the establishment of their nucleotide sequence, the presence of RK2 oriT, and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of the trfA gene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of the Acetobacter xylinum phosphoglucomutase gene celB and the two commonly used reporter genes luc and cat in Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas campestris. Good induction properties and tight regulation of Pm were achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. Induced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and PT7 promoters located on high copy number plasmids. The induced levels of Luc activity were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains other than E. coli. We believe that the well-characterized vectors described here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria.

The STL1 gene of Saccharomyces cerevisiae is predicted to encode a sugar transporter-like protein.

A gene has been cloned from the yeast Saccharomyces cerevisiae which, on the basis of the deduced translation product, encodes a sugar transporter-like protein. This gene, STL1, was identified as an open reading frame (ORF) closely linked to the cinnamic-acid-resistance gene POF1 on chromosome IV. The putative translation product of STL1 (STL1) contains 536 amino acids, with a M(r) of 60,507. Hydropathy analysis of STL1 suggests that it contains the twelve transmembrane (TM) domains characteristic of a family of sugar transporters from S. cerevisiae and other organisms. STL1 displays greatest homology (28% identity) to the products of the yeast HXT2 (hexose transporter) and GAL2 (galactose transporter) genes. Disruption of STL1 had no detectable effect on yeast growth on glucose, galactose, mannose, maltose or glycerol as sole carbon source. The transport function of the gene product remains unknown at present.